Mitochondrial dysfunction is a fundamental problem associated with a significant number of human aging-associated diseases. Various protective strategies targeting mitochondrial dysfunctions are aimed mostly at scavenging or inhibiting generation of toxic reactive oxygen species (ROS). However, these approaches, while partly successful, would benefit from an understanding of other mechanisms leading to aging-related accumulation of dysfunctional mitochondria. A critical question is how mitochondrial quality is maintained/ regulated. We (and others) have recently shown that the ubiquitin/proteasome system controls the functional integrity of the mitochondria. Consistent with this, our data demonstrate that proteins of the outer mitochondrial membrane are modified by ubiquitin conjugation, and their degradation is mediated by the proteasome. Furthermore, we also found that AAA- ATPase p97, a critical protein for the endoplasmic reticulum associated degradation (ERAD) pathway, is essential for both regulation of ubiquitination-dependent turnover of the outer mitochondrial membrane proteins and for autophagy-mediated degradation of functionally compromised mitochondria. These data place p97 in the center of two fundamental catabolic pathways, i) ubiquitin-dependent protein degradation and ii) mitochondria-specific autophagy (mitophagy). Studies proposed here will further scrutinize the mechanisms and scope of the ubiquitin/proteasome system and p97, in particular, in the regulation of mitochondrial homeostasis. Three questions will be addressed: 1) what is the mechanism and scope of p97 in ubiquitin-dependent mitochondrial protein degradation? These studies will include characterization of cofactors acting together with p97 on the mitochondria. We will also ask whether non-OMM mitochondrial proteins are dislocated from the mitochondria in p97-dependent manner. 2) Do ubiquitin/proteasome system and p97 serve as a mitochondrial quality control mechanism? The role for p97 in degradation of mitochondria-associated mutant proteins will be scrutinized. 3) How does p97 activate mitochondrial specific autophagy? The experiments will include analyses of distinct models of how p97 regulates mitophagy. Studies using cultured cells and in vitro assays will be carried out, along with imaging investigations using time- lapse methods and new fluorescent tools, including photoactivated fluorescent proteins and recently developed by us fluorescent ubiquitination chain sensors. Mutagenesis and RNAi methods will also be exploited. PUBLIC HEALTH RELEVANCE: Dysfunctions of mitochondria are linked to numerous debilitating pathologies including Parkinson's disease (PD), Huntington's disease (HD), Alzheimer's disease (AD), amyotrophic lateral sclerosis, and diabetic complications. The goal is to define the role and mechanism of AAA-ATPase p97 with mitochondrial protein ubiquitination in the regulation of mitochondrial function and integration of mitochondrial quality control pathways. We believe that characterizing this process in its molecular detail will lead us to important insights into new aspects of mitochondrial biology, and ultimately to novel therapeutic targets for mitochondria dysfunction-linked diseases.